Morton D. Prager

Asparagine synthetase in normal and malignant tissues; correlation with tumor sensitivity to asparaginase

Abstract Asparagine synthetase activity has been measured in normal and malignant mouse and normal guinea pig tissues. The enzyme has a pH optimum of 8.0, requires ATP, uses either glutamine or NH 4 Cl as a source of amide, and yields increased amounts of asparagine in the presence of puromycin. Significantly higher ASase activity has been observed in four asparaginase resistant mouse lymphomas than in two susceptible tumors or in normal mouse tissue. Liver is an exception, but exact values are rendered uncertain by the presence of asparaginase. When asparaginase is administered to tumor-bearing mice, resistant tumors respond with large increases (5 to 19-fold) in ASase activity over a 24-hr period while susceptible tumors respond only transiently, ASase levels reaching a maximum in 4 hr and then decreasing concomitantly with cell death. These observations confirm and extend the idea that ASase activity may provide a basis for tumor resistance or sensitivity to asparaginase. The moderate increases in ASase in normal tissue following asparaginase administration account for the low toxicity of the enzyme. The levels reached in normal mouse tissue are equivalent to those of the tissues of guinea pigs which have asparaginase in the circulation as a natural constituent. The increased ASase activity following asparaginase administration may be accounted for by contributions of both reduced product inhibition and derepression of ASase synthesis. Induction apparently plays no role in these tumors. ASase appears in the postmicrosomal supernates of tumor cells and to a lesser degree in microsomes. In liver essentially all of the detectable ASase is in the microsomes, and asparaginase is present in the supernate.

Asparagine synthetase in asparaginase resistant and susceptible mouse lymphomas.

Abstract Since the original discovery of a tumor inhibitory factor in guinea pig serum by Kidd (1953) and its identification as L-asparaginase by Broome (1961) , this enzyme has been used to regress tumors, especially lymphomas, of the mouse, rat, dog, and man. The work of Sobin and Kidd (1966) showed that protein synthesis was the first biochemical system to be inhibited by asparaginase treatment of a susceptible tumor. Broome and Schwartz (1967) reported that resistant tumors have a greater endogenous supply of asparagine. Furthermore, Patterson and Orr (1967) found that an asparaginase resistant form of the Jensen sarcoma had a greater capacity for asparagine synthesis than either a susceptible line or normal liver. The present studies show that asparaginase resistant lymphomas have higher levels of asparagine synthetase (ASase) than either susceptible tumors or normal tissue. In addition, resistant tumors responded to asparaginase treatment with large increases in ASase levels, a moderate response was noted for normal tissue, and a susceptible tumor gave only a transient response.

Fabrication of resorbable microporous intravascular stents for gene therapy applications.

The authors have produced resorbable, microporous endoluminal stents from Poly-L-lactic acid (PLLA)/Poly epsilon-caprolactone (PCL) blends. Both helical and tube stent designs have been obtained by solvent casting and flotation-precipitation fabrication techniques. A range of PLLA/PCL blend ratios and process variables were employed to investigate their influence on mechanical properties, porosity, and degradation rate. Polymer blends with higher PLLA proportions exhibit higher elastic moduli and ultimate tensile strength, and lower elongation, porosity, and degradation rates than do materials with higher PCL content. Stents with suitable mechanical properties for deployment and support of the vessel wall were obtained. Poly(ethylene oxide) was incorporated into these devices using an acid swelling technique, opening the pore structure and improving the hydrophilic character, thereby enabling the uptake of recombinant adenoviral vectors. The 50:50 PLLA/PCL blended stents were impregnated with recombinant adenovirus (AdCMB beta Gal, encoding a nuclear localizing variant of Escherichia coli beta-galactosidase). Cultured CV-1 cells incubated with stents impregnated with the recombinant virus expressed nuclear localized beta-galactosidase activity, confirming that absorbed virus is released from the matrix in an infectious form, with kinetics suggesting that genetically enhanced endovascular devices of this design are feasible.

The enhancement of humoral and cellular immune responses by dimethyldioctadecylammonium bromide

Abstract Dimethyldioctadecylammonium bromide (DDA) produced marked enhancement of both cellular and humoral immune responses to SRBC when administered to mice intraperitoneally, or of cellular immunity when given subcutaneously. Stimulated cellular responses were seen as increased footpad swelling as a measure of delayed hypersensitivity and increased antigen-induced blastogenesis. Elevation of humoral response was reflected in increased numbers of splenic plaque-forming cells (PFC) and in circulating anti-SRBC antibody. Adjuvancy did not depend on addition of the lipid of DDA to antigen, as both humoral and cellular responses were enhanced whether DDA and SRBC were admixed or injected separately 4 hr apart intraperitoneally. DDA also enhanced the PFC response to the T-cell independent antigen TNP-LPS. The DDA effects are accompanied by macrophage activation, which may mediate at least in part the observed responses. DDA-activated macrophages exhibit fast spreading, are highly phagocytic, and elaborate significantly greater amounts of thymocyte mitogenic factor(s) than do normal resident peritoneal macrophages. This activation may effect the stimulation of antigen-specific primary lymphocyte responses by adjuvant and expansion of memory-cell populations which lead to the observed enhancement of secondary responses.

Radiochemical studies on contact lens soilation. I. Lens uptake of 14C-lysozyme from simple and complex artificial tear solutions

A systematic study has been made of the uptake of lysozyme by various contact lens materials. Lens uptake of 14C-methylated lysozyme was assessed using simple to complex artificial tear solutions. The data reflect prominent uptake by Group IV (ionic, high-water-content) hydrogel lenses, consistent with the literature. This includes protein on the lens surface and in the lens matrix, the former being estimated at about 33% of the total deposit for the DuraSoft 3 lenses used. Uptake appears to be contingent upon the nature of the lens material, the composition of the deposit model used, and the duration of lens exposure to the artificial tear solution.

Effect of melatonin on activated macrophage TNF, IL-6, and reactive oxygen intermediates.

ABSTRACT Melatonin has recently been investigated as a biological response modifier in sepsis and hypovolemic shock. Although melatonin is reported to influence a variety of inflammatory and immune responses, evidence supporting its effects on important macrophage-derived mediators is incomplete. This study was designed to determine whether melatonin alters the release TNF, IL-6, and reactive oxygen intermediates by activated macrophages. TNF and IL-6 bioactivity in LPS-stimulated Wistar rat alveolar macrophage and RAW 264.7 cell culture supernatants were unchanged by pretreatment with melatonin. Similarly, macrophage production of reactive oxygen intermediates, including H2O2 and superoxide anion, were unaffected by melatonin pretreatment. PMA-stimulated H2O2 production was determined in rat alveolar macrophages and RAW 264.7 cells. Superoxide anion generation was determined in the rat alveolar macrophage NR8383 cell line. Melatonin, at concentrations ranging from 10-7 to 10-4 M, does not alter LPS-stimulated TNF and IL-6, or PMA-stimulated H2O2 and superoxide anion production by the macrophage populations studied. These observations are in contrast to previous reports. Further studies are necessary to determine whether melatonin indirectly influences macrophage function by actions on nonmacrophage cell populations.

Chitosan and chitosan sulfate have opposing effects on collagen–fibroblast interactions

Soon after injury, hyaluronan is prominent in granulation tissue. As hyaluronan wanes, sulfated glycosaminoglycans predominate. The temporal relationship between the transition from unsulfated to sulfated glycosaminoglycans and the phenotypic changes in fibroblasts in the wound bed suggest that these two events are interrelated. This possibility was investigated using chitosan and its sulfated product as model compounds. The ability of cultured human foreskin fibroblasts to bind and to contract lattices of collagen, collagen–chitosan, and collagen–chitosan sulfate was determined. Fibroblast adherence to substrates after 24 hours was determined by the MTT assay at A570. Adherence to the collagen–chitosan substrate was markedly reduced (mean A570± SD; 0.16 ± 0.05, n = 6) (p < 0.01) compared to collagen alone (0.92 ± 0.04) or to collagen–chitosan sulfate (0.84 ± 0.05). Kinetics of contraction of lattices by enmeshed fibroblasts was determined by planimetric measurements, 0–48 hours after loosening the lattices. Contraction of the collagen–chitosan lattices (n = 5) was less at all time points than for the other two lattices. After 48 hours, the collagen– chitosan lattices contracted significantly (p < 0.01) less (30.0%± 4.4) compared to collagen alone (66.9%± 4.7) and collagen–chitosan sulfate (71.6%± 7.7). Scanning electron microscopy of the acellular lattices showed fibers of the collagen–chitosan mixture to be the thickest and with altered organization. These results show that chitosan sulfation markedly enhances fibroblast adhesion and promotes contraction of a collagen lattice compared to the unsulfated material. By analogy to the in vivo sequence of hyaluronan followed by sulfated glycosaminoglycans in wounds, the results suggest that glycosaminoglycan sulfation may be a contributing signal for phenotypic transformation during wound healing.

Radiochemical studies on contact lens soilation. II. Lens uptake of cholesteryl oleate and dioleoyl phosphatidylcholine

Employing an artificial tear preparation composed of six proteins and six lipids as a deposit model, uptake of the lipids 3H-cholesteryl oleate and 14C-dioleoyl phosphatidylcholine was measured on contact lenses representative of the four FDA hydrogel groups and on select RGP lenses. Cholesteryl oleate uptake after 24 h at 37 degrees C generally was less than 1 microgram/lens although occasionally reaching 1-2 micrograms. DuraSoft 3 lenses (Group IV) accumulated the deposits in greater amounts (p = 0.04) with other lens groups not differing significantly from each other. Ionic DuraSoft 2 and 3 lenses bound more phosphatidylcholine (also < 1 microgram) than other lens groups, possibly reflecting an interaction between the positively charged choline residue and the negative surface of the lens. Lysozyme deposition, measured simultaneously with cholesteryl oleate, bound to a far greater extent to Group IV lenses (e.g., DuraSoft 3, mean surface deposit 279 micrograms) than to other lens types (p < 0.01). Multiple application of the artificial tear solution did not produce a statistically significant increase in cholesteryl oleate accumulation.

Anti-platelet action of nitric oxide and selective phosphodiesterase inhibitors.

Nitric oxide gas is a potent inhibitor of platelet aggregation, with an IC50 of 3.6 μM for rabbit platelets. Since the NO effect is mediated via increased cGMP, this in vitro study was undertaken to test the hypothesis that selective phosphodiesterase (PDE) inhibitors might enhance aggregation inhibition at lower NO concentrations. Because the cAMP-selective PDE III and the cGMP-selective PDE V are prominent in platelets, milrinone, a PDE III inhibitor, and zaprinast, a PDE V inhibitor, were tested alone and in the presence of NO for their effect on aggregation. Aggregometry was performed on rabbit platelet-rich plasma following addition of ADP as agonist. Milrinone alone gave an IC50 of 12.4 μM. With each agent set to give suboptimal inhibition of aggregation, the combination of milrinone (3–16 μM) and NO (2–10 μM) produced a greater effect than either agent alone. Zaprinast exhibited no effect on aggregation in concentrations up to 160 μM. However, adding zaprinast to 2 μM NO, which alone reduced aggregation ∼30%, produced a marked synergism in the inhibitory effect up to and including no observable aggregation. These results indicate that elevation of either cAMP or cGMP is sufficient to inhibit platelet function. The platelet cAMP concentration appears high enough to be inhibitory when degradation is suppressed by milrinone. However, basal cGMP levels must be increased by NO before the zaprinast effect is observed.

Proteolytic activity in burn wound exudates and comparison of fibrin degradation products and protease inhibitors in exudates and sera

Proteolytic (caseinolytic) activity in burn wound exudates was screened over the range pH 5.3 to 8.4. Although activity was greatest at pH 8.4 in four of seven exudates, individual differences indicated that different proteases predominate in the local environment of the wound. Paired exudate and serum samples were compared with regard to fibrin degradation products and three protease inhibitors: antithrombin III, a1-protease inhibitor, and a2-antiplasmin. Fibrin degradation products concentration was higher in exudates than in paired sera, indicating the wound as the source of circulating fibrin degradation products rather than intravascular coagulation followed by fibrinolysis. In contrast, all three protease inhibitors exhibited higher concentrations in serum than in the paired exudate. The serum/exudate ratio for AT III differed significantly from that for a1-protease inhibitor and a2-antiplasmin, and the ratio of two inhibitors in serum differed from the ratio of the same two inhibitors in the exudate in two of three comparisons. These findings emphasize the importance of exudate examinations as a reflection of events in the wound itself. The importance of microenvironments is invoked to account for the significant exudate fibrin degradation products titers, which are seen despite the presence of antithrombin III, which could inhibit coagulation, and the presence of a2-antiplasmin, which could inhibit fibrin degradation.